EP0731808A1 - Nucleosides et oligonucleosides contenant des amas de bore - Google Patents

Nucleosides et oligonucleosides contenant des amas de bore

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Publication number
EP0731808A1
EP0731808A1 EP95904208A EP95904208A EP0731808A1 EP 0731808 A1 EP0731808 A1 EP 0731808A1 EP 95904208 A EP95904208 A EP 95904208A EP 95904208 A EP95904208 A EP 95904208A EP 0731808 A1 EP0731808 A1 EP 0731808A1
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Prior art keywords
oligonucleotide
carboran
carboranyl
methyl
oligonucleotides
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EP95904208A
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German (de)
English (en)
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EP0731808A4 (fr
EP0731808B1 (fr
Inventor
Raymond F. Schinazi
Geraldine F. Kattan
Zbigniew J. Lesnikowski
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Emory University
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Emory University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/009—Neutron capture therapy, e.g. using uranium or non-boron material
    • A61K41/0095—Boron neutron capture therapy, i.e. BNCT, e.g. using boronated porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/33—Chemical structure of the base
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3527—Other alkyl chain

Definitions

  • This invention is in the area of synthetic organic chemistry, and is in particular carboranyl containing synthetic nucleosides and oligonucleotides, and their method of preparation and use.
  • the goal of cancer therapy is to achieve a degree of selectivity that spares normal cells and destroys all malignant ones, since even a small number of remaining malignant cells can lead to recurrence, metastasis, and death.
  • a two-component or binary system comprised of constituents that alone are nonlethal and largely confined to malignant cells, and which when combined are lethal to the neoplastic cells yet innocuous to normal cells is an ideal modality.
  • One advantage of this type of binary system is that each component can be manipulated independently to maximize selectivity.
  • Boron neutron capture therapy (BNCT, see Figure 1) is a binary system which combines two separately nonlethal constituents, a radiosensitizing compound that contains a stable boron-10 ( 10 B) isotope, and nonionizing neutron radiation.
  • boron-10 is irradiated with neutrons, a nuclear reaction occurs that yields helium nuclei ( ⁇ -particle) , lithium nuclei, and about 100 million times more energy than the initial irradiated energy.
  • the generated radiation destroys malignant cells containing the boron compound.
  • Selectivity is achieved through the use of compounds which accumulate primarily in malignant cells and/or by aiming the neutron beam at the tumor mass which contains the boron carrier.
  • SUBS ⁇ TUTESHEET(RULE26) The major obstacles in BNCT are: (1) the achievement of a sufficiently high intracellular boron concentration and (2) selectivity toward tumor cells. Although attempts to develop tumor- selective boron compounds date back to the 1960s and despite extensive studies, the problem of selective delivery of boron carriers to tumor cells remains .
  • 5,130,302 to Eisenvogel, et al . discloses a novel class of boronated nucleosides, nucleotides and oligonucleotides for use as antineoplastic, antiinflammatory, and antihypertensive agents.
  • the nucleosides, nucleotides and oligonucleotides are covalently attached to either BH 2 CN, BH 3 , or BH 2 C0 2 R moieties, wherein R is C- to C 18 alkyl .
  • carboranyl pyrimidines have been prepared for use in boron neutron capture therapy.
  • Examples of carboranyl pyrimidines include 5-(3-o- carboranylpropyl-6-methyl-2-thiouracil (compound A) (Wilson, J. G. Pigment Cell Res 1989, 2, 297-303) , 2,4-dichloro-5- (1-o-carboranylmethyl) -6- methylpyrimidine; (compound B) (Reynolds, R. C. ; Trask, T. W. ; Sedwick, W. D. J " Org Chem 1991, 56, 2391-2395) ; and 5-carboranyluracil (compound C) (Goudgaon, N.
  • Examples include 5-carboranyl-2 ' - deoxyuridine (compound D, CDU) , 5-carboranyluridine (compound E, CU) , 5- (1-hydroxymethyl) carboranyl- uridine, and 5- (1-hydroxymethyl) carboranyluridine (compound F, HMCU) .
  • PCT WO 93/17028 filed by Raymond F. Schinazi and Dennis C. Liotta discloses a number of synthetic nucleosides that contain a carboranyl moiety covalently attached to a purine or pyrimidine base, wherein the sugar moiety optionally contains a second heteroatom in the 3 '-position of the ring.
  • Preferred compounds are 2-hydroxymethyl-5- (5- carboranylcytosin-1-yl) -1, 3-oxathiolane (compound G) and 2-hydroxymethyl-5- (5-carboranyluridin-l-yl) - 1, 3-oxathiolane (compound H) .
  • BNCT BNCT
  • oligonucleotides which include cell selectivity (ability to accumulate preferentially in diseased cells) , stability of the chemotherapeutic agent in vivo (resistance against digestion by cellular nucleases and chemical stability) , and transportability (ability of the chemotherapeutic agent to pass easily through cellular membranes)
  • AOT Antisense Oligonucleotide Technology
  • Antisense technology refers in general to the modulation of gene expression through a process wherein a synthetic oligonucleotide is hybridized to a complementary nucleic acid sequence to inhibit transcription or replication (if the target sequence is DNA) , inhibit translation (if the target sequence is RNA) or to inhibit processing (if the target sequence is pre-RNA) .
  • a wide variety of cellular activities can be modulated using this technique.
  • a simple example is the inhibition of protein biosynthesis by an antisense oligonucleotide bound to mRNA.
  • a synthetic oligonucleotide is hybridized to a specific gene sequence in double stranded DNA, forming a triple stranded complex (triplex) that inhibits the expression of that gene sequence.
  • Antisense oligonucleotides can be also used to activate gene expression indirectly by suppressing the biosynthesis of a natural repressor or directly by reducing termination of transcription.
  • AOT can be used to inhibit the expression of pathogenic genes, for example, those that facilitate the replication of viruses, including human immunodeficiency virus (HIV) , hepatitis B virus (HBV) , and herpesviruses, and cancers, particularly solid tumor masses such as gliomas, breast cancer, and melanomas.
  • viruses including human immunodeficiency virus (HIV) , hepatitis B virus (HBV) , and herpesviruses, and cancers, particularly solid tumor masses such as gliomas, breast cancer, and melanomas.
  • Carboranyl-containing nucleosides and oligonucleotides are provided for use in boron neutron capture therapy (BNCT) that are lipophilic and have a high content of boron atoms .
  • BNCT boron neutron capture therapy
  • dinucleotides and oligonucleotides are provided that contain at least one uncharged 3', 5'- 0,0- [ (carboran-1-yl-methyl)phosphonate] internucleotide linkage in place of the naturally occurring 3 ' , 5' -0,O-phosphodiester residue.
  • the (carboran-1-yl-methyl)phosphonate linkage is not degraded by nucleases, and therefore, dinucleotides and oligonucleotides that contain the 3 ',5' -0,0- [ (carboran-l-yl-methyl)phosphonate] internucleotide linkage are stable in biological fluids and cells.
  • oligonucleotides are provided in which the two terminal nucleosides at the 3'- end, or nucleosides adjacent to these nucleosides, are linked via the nuclease-stable 3 ' , 5' -0,0- [ (o- carboran-1-yl-methyl)phosphonate] bridge.
  • the 3 ' , 5' -0,0- [ (o-carboran-l-yl-methyl)phosphonate] bridge is also stable in acidic environments and is highly thermally stable.
  • Oligonucleotides can be designed for BNCT according to methods described herein that are complementary to overexpressed or unique RNA or DNA sequences in target cancer cells, as a means to selectively accumulate the boron-containing material into these cells. Oligonucleotides of specific gene sequences that include one or more 3' , 5' -linking- (carboran-1-yl)phosphonate moieties can also be used in antisense therapy in the selective modification of gene expression. In a second embodiment, nucleosides are provided that bear a (carboran-l-yl)phosphonate moiety in the 3' and/or 5' -position. These synthetic nucleotides are useful in boron neutron capture therapy, and selected compounds can exhibit activity against viruses such as HIV and HBV.
  • oligonucleotides are provided that bear a carboranyl-modified base in at least one of the nucleosides of the oligomer.
  • the carboranyl-containing base is in a nucleoside located at the 3' -terminus, in the nucleoside adjacent to the 3 '-terminal nucleoside, in the 5' -terminal nucleoside, or in the nucleoside adjacent to the 5' -terminal nucleoside.
  • Oligonucleotides bearing carboranyl- containing bases in the 3 '-terminal nucleoside or the nucleoside adjacent to the 3' -terminal are more resistant to degradation by 3 ' -exonucleases.
  • oligonucleotides bearing carboranyl-containing base units in the preferred positions hybridize more effectively to complementary nucleic acid sequences than oligonucleotides bearing carboranyl-containing bases in other positions.
  • oligonucleotides are provided that bear at least one 3',5'-[(0,0- carboran-1-yl) phosphonate] residue and at least one nucleoside that contains a carboranyl- containing base, as a means to increase the boron density and lipophilicity of the molecule, and depending on the location of the modifications, increase the stability of the oligomer in vivo in biological fluids or cells.
  • the oligomers disclosed herein can be used in vitro to carry out structure- activity relationships on the bulk tolerance of hybridization of synthetic oligonucleotides with complementary nucleic acid sequences, in MRI imaging, or as probes in a variety of diagnostic techniques.
  • Carboranyl-containing oligonucleotides can also be used to effect mutation of expressed HIV-1 reverse transcriptase, using in vitro or in vivo Site Directed Mutagenesis (SDM) .
  • Nucleosides, nucleotides, and oligonucleotides can be prepared that contain boron clusters as a means to enhance lipophilicity wherein the boron is not enriched in 10 B, but instead, in the ⁇ B isotope.
  • nucleosides, nucleotides, and oligonucleotides of the present invention that are used for BNCT or other diagnostic techniques that depend on neutron radiation decay for the destruction of diseased cells or for signaling purposes should be enriched with a suitable amount of 10 B, normally approximately 90-100% 10 B, and typically between 92- 96% 10 B.
  • a novel process is provided for the preparation of nucleosides, dinucleotides, and oligonucleotides containing an (carboran-l-yl-methyl)phosphonate moiety via the key starting material 0- methyl (carboran-l-yl)methyl phosphonate.
  • Figure 1 is a schematic illustration of a hypothetical mechanism of action of boronated oligonucleotides for BNCT.
  • Figure 2 is a schematic illustration of a process for the preparation of thymidine- (3 ' , 5') - thymidine (o-carboran-1-yl) methylphosphonate using the key starting material O-methyl (o-carboran-1- yl) methyl phosphonate.
  • Figure 3 is an illustration of a process for the preparation of O-methyl- [0- (o-carboran-1- yl) lkyl] phosphate.
  • Figure 4 is an illustration of a process for the preparation of 5- (o-carboranyl) -5' -O- dimethoxytrityl-2' -0-deoxyuridine-3' - (N,N- diisopropyl- ⁇ -cyanoethyl)phosphoramidite.
  • Figure 5 is an illustration of the chemical structures of a B,_H 10 carborane moiety and the anionic o-ni o-7, 8-C 2 B 9 H (11 or 12) and o - cl oso- 1, 2-C 2 B 10 H 12 forms of carborane.
  • Figure 6 is an illustration of a process for the automated preparation of dodecathymidylic acid analogues bearing one or more 5- (o-carboran-1- yDuracil residues, as further described in Table
  • Figure 8 is the circular dichroism spectra of selected single stranded CDU modified and unmodified d(T) 12 (compound 18) .
  • Figure 9 is the circular dichroism spectra of complexes formed between CDU modified and unmodified d(T) 12 and d(A) 12 .
  • alkyl refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon of C, to C 10 , and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2- dimethylbutyl, and 2, 3-dimethylbutyl .
  • the alkyl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al . , "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991.
  • the term lower alkyl, as used herein, and unless otherwise specified, refers to a C, to C 4 saturated straight or branched alkyl group.
  • alkylamino or arylamino refers to an amino group that has one or two alkyl or aryl substituents, respectively.
  • protected refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes.
  • oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
  • aryl refers to phe yl, biphenyl, or naphthyl, and preferably phenyl .
  • the aryl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl, amino,' alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al . , "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991.
  • alkaryl or alkylaryl refers to an alkyl group with an aryl substituent.
  • aralkyl or arylalkyl refers to an aryl group with an alkyl substituent.
  • halo includes chloro, bromo, iodo, and fluoro.
  • purine or pyrimidine base includes, but is not limited to, adenine, JN ⁇ -alkylpurines, N 6 - acylpurines (wherein acyl is C(0) (alkyl, aryl, alkylaryl, or arylalkyl) , IS ⁇ -benzylpurine, N 6 - halopurine, IN ⁇ -vinylpurine, ⁇ -acetylenic purine, N 6 - acyl purine, N ⁇ -hydroxyalkyl purine, ⁇ -thioalkyl purine, N 2 -alkylpurines, N 2 -alkyl-6-thiopurines, thymine, cytosine, 6-azapyrimidine, 2- and/or 4- thiopyrmidine, uracil, C 5 -alkylpyrimidines, C 5 - benzylpyrimidines, C 5 -halopyrimidines, C 5 - vinylpyrimidine, C 5 -acetyle
  • Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t- butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl, and p- toluylsulfonyl .
  • heteroaryl or heteroaromatic refers to an aromatic moiety that includes at least one sulfur, oxygen, or nitrogen in the aromatic ring.
  • Nonlimiting examples are furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl; benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4- thiadiazolyl, isooxazolyl, pyrrolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, and pteridinyl .
  • Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t- butyldimethylsilyl, and t-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl, and p-toluylsulfonyl .
  • alkenyl refers to a straight, branched, hydrocarbon of C 2 to C 10 with at least one double bond.
  • acyl refers to moiety of the formula - C(0)R', wherein R' is alkyl; alkoxyalkyl including methoxymethyl; arylalkyl including benzyl; aryloxyalkyl such as phenoxymethyl; aryl including phenyl optionally substituted with halogen, C x to C 4 alkyl or C- to C 4 alkoxy, or the residue of an amino acid.
  • enantiomerically enriched refers to a compound that is a mixture of enantiomers in which one enantiomer is present in excess, and preferably present to the extent of 95% or more, and more preferably 98% or more, in the mixture.
  • oligonucleotide refers to an oligomer of thirty-five or less nucleotides linked through their 3' and 5' -hydroxyl or 2' - and 5' -hydroxyl groups .
  • amino acid includes naturally occurring and synthetic a .no acids, and includes but is not limited to, alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, and histidinyl . It should be understood that when the term
  • (carboran-l-yl)phosphonate is used in this text, it should be understood that -O- (carboran-l- yl) alkyl] phosphate, S- (carboran-l- yl) alkyl] phosphorothioate, or Se- (carboran-1- yl) alkyl] phosphoroselenoate can be used in place thereof .
  • a new class of modified lipophilic nucleotides and oligonucleotides bearing carboranyl residues is provided for use in BNCT, AOT and for diagnostic purposes, including an MRI and as probes.
  • the oligonucleotides bear one or more carboran-l-yl residues, allowing for the concentrated and selective administration of boron to target cells.
  • the lipophilicity of boron-modified oligonucleotides can be manipulated by the appropriate selection of the number and location of the carboran-l-yl residues in the compound.
  • a carboranyl group linked directly to a phosphorus atom or through an appropriate spacer (e.g., alkyl, peptidyl) attached to oxygen, sulfur, or selenium has a more significant effect on the lipophilicity of the compound than when the carboranyl group is attached in another location, such as on the base, because it acts as a substitute for a hydrophilic and ionizable hydroxy group.
  • the carboranyl-containing oligonucleotides are targeted specifically to cancer cells to inhibit the overexpression of certain protooncogenes or to optimize expression of tumor suppressor genes which correlate well with clinical progression of tumors, including gliomas, melanomas, and breast tumors.
  • Carboranes also referred to as carbaboranes are compounds that contain carbon atoms incorporated into a polyhedral borane.
  • the CH group is isoelectronic with BH", and thus can replace a BH " group.
  • Polyhedral carboranes can thus be considered as formally derived from B n H n.2 ions, with two carbon replacements, leading to molecules of the general formula B n . 2 C 2 H n .
  • Neutral two carbon carboranes are generally of the formula B n C 2 H n+2 , wherein n is 3-10.
  • the carborane is referred to as a 1,2- or ortho-carborane (o- carborane) .
  • B 10 C 2 H 12 is usually prepared as a 1,2-isomer, which when heated rearranges to a 1,7-isomer.
  • Carboranes can exist in a number of isomeric forms.
  • “Closo” carboranes are closed cage structures, whereas “nido” carboranes are open nest-like structures. Examples are anionic o-nido- 7, 8-C 2 B 9 H ⁇ ior 12) and neutral o- closo- 1 , 2-C 2 B 10 H 12 .
  • Carbaboranes can also exist as one of four arachno isomers or as a hypho-isomer. Both the 1,2- and the 1, 7-dicarbadodecaboranes and their C- substituted derivatives, on treatment with strong base, are degraded with loss of boron to give isomeric nido-carborane anions, B 9 C 2 H (11 or 12) " . Both isomeric B 9 C 2 H (n or I2) " ions on treatment with anhydrous acid followed by heating are converted into the closo-carborane B 9 C 2 H n . Carbaboranes are typically prepared by the interaction of boranes or borane adducts with acetylenes .
  • the most common carboranes are B 10 C 2 H 12 and its carbon-substituted derivatives.
  • Carbon- substituted carboranes can be prepared with substituted acetylenes, as known to those skilled in the art, or, for example, by reaction of the carbaborane with a strong base to replace a hydrogen with lithium, followed by treatment with a desired electrophilic reagent.
  • Acetylene derivatives that can be used to provide substituted carborane moieties are described, for example, in Heying, T. L . , et al . Inorganic Chemistry 2(6) 1089- 1092, 1963) .
  • Anionic carboranes can be administered as a pharmaceutically acceptable salt of a single or multivalent pharmaceutically acceptable cation, including but not limited to, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, pyridinium, quaternary amine, ammonium, protonated ethylenediamine, or protonated amino acids, including but not limited to protonated lysine and protonated arginine .
  • a pharmaceutically acceptable salt of a single or multivalent pharmaceutically acceptable cation including but not limited to, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, pyridinium, quaternary amine, ammonium, protonated ethylenediamine, or protonated amino acids, including but not limited to protonated lysine and protonated arginine .
  • a nucleoside is provided that contains an (carboran-l-yl-methyl)phosphonate in the 3' or 5' -position of the molecule.
  • Nonlimiting examples are the nucleosides of Formulas I, II, and III illustrated below:
  • R 1 is alkyl, haloalkyl, alkenyl, alkoxyalkyl, aryl, heteroaryl, trifluoromethyl, alkylaryl, arylalkyl, or halogen;
  • R 2 is hydrogen, alkyl, acyl (including acetyl) ; sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl; a mono, di or triphosphate ester; trityl or monomethoxytrityl; benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of aryl given above; silyl, including trialkylsilyl (e.g.
  • R 3 is hydroxyl, hydrogen, halogen, -CN, -N 3 , lower alkyl, amino, alkylamino, dialkylamino, alkoxy; and wherein the R 3 group can be in the ribosyl ("down" with respect to the sugar moiety when orienting the ring such that the oxygen is in the back) or the arabinosyl ("up") conformation;
  • B represents the boron moiety of a carboranyl group, and specifically includes anionic o-nido-7, 8-C 2 B 9 H (llorl2) and neutral o- closo-l J -C 2 B 10 H 12 ;
  • W is O, S, or Se;
  • Z is O or S;
  • X is O, S, S(O) , S(0) 2 , CH 2 , CHOH, CHN 3 or
  • Y is OH, SH, SeH, or halogen, and in particular, fluorine; n is 1-5; and m is 0 or 1.
  • the base is preferably a purine or pyrimidine base as defined above, and preferably is thymine, uracil, 5-halouracil including 5-fluorouracil, cytosine, 5-halocytosine including 5- fluorocytosine, adenine, guanine, 2,6- diaminopurine, 2-amino-6-chloropurine, 2- aminopurine, 5-lower alkyl uracil, or 5-lower alkylcytosine, 2-thiouracil, 2,4-thiouracil, 4- thiouracil, 6-chloropurine, 5-carboranyluracil, 5- carboranylcytosine and other carboranylpurines and carboranylpyrmidines, including those described in Section iv) below.
  • a dinucleotide wherein two nucleosides are connected via an uncharged 3 ', 5' -0,0- [(carboran-l-yl- methyl)phosphonate] linkage.
  • Nonlimiting examples are compounds of Formulas III and IV:
  • R 1 , R 2 , R 3 , B, W, X, Y, Z, m and n are as defined above.
  • oligonucleotides and phosphothioate or dithioate oligonucleotides, methylphosphonate oligonucleotides, and dephosphooligonucleotides are provided that contain at least one 3' , 5' -0,0- [ (carboran-l-yl- methyl)phosphonate] internucleotide linkage.
  • the 3 ' , 5' -0,0- [ (carboran-l-yl-methyl)phosphonate can link the terminal two nucleotides at the 3'-end of the oligonucleotide, the terminal two nucleotides at the 5' -end of the oligonucleotide, or, alternatively, two nucleotides in the internal section of the oligonucleotide, including adjacent ones.
  • an oligonucleotide is provided wherein a 3', 5' -0,0- [(carboran-l-yl- methyl)phosphonate links at least the terminal two nucleotides at the 3'-terminus or the nucleosides adjacent to these.
  • the oligonucleotide if desired, can contain more than one 3 ', 5' -0,0- [(carboran-l-yl- methyl)phosphonate linkage, up to a fully modified oligonucleotide.
  • the oligonucleotide has between approximately one and five modified linkages for a typical (thirty or less) -mer.
  • any of the purine or pyrimidine bases defined above can be used in the oligonucleotide, in any appropriate sequence, as discussed in more detail
  • nucleosides such as adenosine, guanosine, cytidine, thymidine, or uridine, are present in the oligonucleotide.
  • a nucleotide can be used as the 3 '-terminus that contains an X moiety, wherein X is 0, S, S (0) , S(0) 2 , CH 2 , or NH, and preferably 0 or S .
  • Synthetic oligonucleotides with 3 ',5' -0,0- [(carboran-l-yl-methyl)phosphonate] linkages of specific sequences can be prepared for hybridization to a complementary nucleic acid sequence to inhibit transcription or replication (if the target sequence is DNA) inhibit translation (if the target sequence is RNA) , or to inhibit processing (if the target sequence is pre-RNA) .
  • Antisense carboranyl-modified oligonucleotides can be prepared, for example, that inhibit protein biosynthesis by hybridization to a target mRNA sequence, and for other purposes as described in the Background of the Invention.
  • Carboranyl-containing oligonucleotides can also be prepared that hybridize to a specific gene sequence in double stranded DNA to form a triple stranded complex (triplex) that inhibits the expression of that gene sequence.
  • nucleic acid sequences with known function have been reported, and given the extensive research currently being conducted in this area, many others will be reported in the future. Given the disclosure herein, one of ordinary skill in the art can prepare any nucleic acid sequence modified as desired with one or more 3 ' , 5' -0,O- [ (carboran-l-yl-methyl)phosphonate] linkages for use in BCNT or AOT. It should be understood that this invention is not directed to specific nucleic acid sequences, but instead is a general technique to increase the stability, lipophilicity, transportability, and boron concentration of a sequence of interest.
  • oligonucleotides are provided that contain a carboranyl moiety in a base unit of one of the nucleotides.
  • carboranyl containing bases are illustrated in Formulas V through X.
  • C is a carboranyl group such as
  • R 4 is -H, -OH, -CH 2 OH, -CH 2 X (wherein X is halogen) or -B 9 C 2 H (Uor 12) (a nido- carborane anion) ;
  • R 5 is lower alkyl; G is N or CH; M is 0 or S; and z is 0 to 5.
  • the carboranyl-containing base can be in a 3'- or 5' -terminal nucleotide, in a nucleotide adjacent to the 3' or 5' -terminal nucleoside, or in an internal nucleotide. It has been discovered that oligonucleotides that contain carboranyl-modified bases in the 3'- or 5' -terminal nucleotide or in a nucleotide adjacent to the 3'- or 5' -terminal nucleoside hybridize more effectively to complementary nucleic acid sequences than oligonucleotides that bear carboranyl-containing bases in internal nucleotides. It has also been discovered that oligonucleotides that contain carboranyl-modified bases in the 3'-terminal nucleotide, in a nucleotide adjacent to the 3' -
  • any nucleic acid sequence of interest can be modified by addition of a carboranyl moiety to a base unit in the oligomer.
  • This invention is not directed to specific nucleic acid sequences, but instead is a general technique.
  • G/Z-2/AS 5 ' -GACACCCAATTCTGAAATGG-3 ' (as G/Z-1/CDU/AS but unmodified)
  • G/Z-3/R 5' -GCACCCATCGACGTCCAACC-3' random sequence, Genejockey version 1.0 Biosoft
  • G/Z-l/S 5' -CAATTTCAGAATTGGGTGTA-3' sequence complementary to G/Z-l/CDU/AS
  • X is a nucleoside that contains a base illustrated in Section iv) .
  • the X represents an unmodified nucleotide such as thymidine, cytidine, adenosine, guanosine, or uridine, or its corresponding 2' -deoxynucleoside, and the above- identified sequences are modified instead by substitution of a 3 ', 5' -0,0- [ (carboran-l-yl- methyl) phosphonate linkage for a phosphodiester linkage (preferably at or close to the 3'- terminus) , with or without base-carboranyl modification.
  • oligonucleotides that contain both a 3', 5' -0,0- [ (carboran- l-yl-methyl)phosphonate] internucleotide linkage and a carboranyl-containing base.
  • the carboranyl- containing base can be on the same or different nucleotide than that linked via a 3',5' -0,0- [ (carboran-l-yl-methyl)phosphonate] bridge.
  • the stereochemistry of the nucleotides and oligonucleotides presented herein is influenced by the configuration of the nucleosides and the configuration of the chiral (carboran-l-yl- methyl)phosphonate moiety, if present in the compound.
  • the oligonucleotides of the present invention are comprised of naturally occurring nucleosides, preferably adenosine, guanosine, cytidine, thymidine, and uridine that have been modified by addition of a 3',5' -0,0- [(carboran-l-yl-methyl)phosphonate] linkage or by addition of a carboranyl moiety to one or more of the base units.
  • the naturally occurring nucleosides have one stereochemical configuration that is set by nature. However, if a non-naturally occurring nucleoside is used in a oligonucleotide or alone, stereochemical issues become relevant.
  • the 1' and 4' carbons of the sugar or modified sugar moiety (referred to below generically as the sugar moiety) of the synthetic nucleosides are chiral, their nonhydrogen substituents (CH 2 OR 2 and the pyrimidine or purine base, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system.
  • the four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the "primary" oxygen (that between the Cl' and C4' -atoms) is in back) : cis (with both groups “up”, which corresponds to the configuration of naturally occurring nucleosides) , cis (with both groups “down”, which is a non-naturally occurring configuration) , trans (with the Cl' substituent "up” and the C4' substituent "down”) , and trans (with the Cl' substituent "down” and the C4' substituent "up”) .
  • “D-nucleosides” are cis nucleosides in a natural configuration and the "L-nucleosides” are cis nucleosides in the nonnaturally occurring configuration.
  • nucleosides can be used in oligonucleotides or alone in any of these configurations. It is known that certain synthetic nucleosides can be more active, or less toxic, or both, in one configuration than in other configurations. One of ordinary skill in the art given this disclosure can easily determine the optimal stereochemical configuration for a specific synthetic nucleoside for a desired application. Alternatively, the nucleoside can be used as a racemic mixture or in the form of an enantiomerically enriched composition.
  • Enzymatic methods for the separation of D and L enantiomers of cis-nucleosides are disclosed in, for example, Nucleosides and Nucleotides, 12(2) , 225-236 (1993) ; European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and PCT Publication Nos. WO 91/11186, WO 92/14729, and WO 92/14743 filed by Emory University.
  • acylated or alkylated racemic mixture of D and L enantiomers of cis-nucleosides can also be accomplished by high performance liquid chromatography with chiral stationary phases, as disclosed in PCT Publication No. WO 92/14729. and ⁇ -L-Nucleosides can be prepared from methods disclosed in, or standard* modifications of methods disclosed in, for example, the following publications: Jeong, et al . , J. of Med. Chem., 36 , 182-195, 1993; European Patent Application Publication No- 0 285 884; Genu-Dellac, C, G. Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, and
  • Wilson, L.J., et al . "A general method for controlling glycosylation stereochemistry in the synthesis of 2' -deoxyribose nucleosides," Tetrahedron Lett. 1815 (1990) ; and Wilson, L.J., et al . , "The synthesis and anti-HIV activity of pyrimidine dioxolanyl nucleosides," Bioorg. Med. Chem. Lett. 3:2 169-174 (1993) .
  • P-stereodefined, P-chiral oligonucleotides can be prepared using following methods.
  • the influence of absolute configuration at phosphorus of P-chiral antisense oligonucleotides on their physicochemical and biochemical properties has been studied.
  • the absolute configuration at the phosphorus atom affects, among other things, solubility, transportability through cellular membranes, affinity toward complementary sequence of target nucleic acid (melting temperature) , and resistance towards nucleolytic enzymes (Uhlman, et al., Chem . Rev. 1990, 90, 544-584) .
  • a novel method is provided for the preparation of nucleosides containing a 3'-0-[(o- carboran-1-yl-methyl)phosphonate] moiety and dinucleotides that contain a 3 ' , 5' -0,0- [ (o- carboran-1-yl-methyl)phosphonate] linkage.
  • the method involves the use of the key starting material, O-methyl (o-carboran-1-yl)methyl phosphonate, a new and versatile borophosphonylating agent.
  • O-methyl (o- carboran-l-yl)methyl phosphonate can be prepared in a three step procedure.
  • propargyl bromide is reacted with trimethyl phosphite in a Michaelis-Arbuzuv type reaction to yield 0,O-dimethylpropargylphosphonate in good yield.
  • Propargyl bromide can be obtained from
  • reaction solvent Aldrich Chemical Company as a solution in toluene, and toluene is used as the reaction solvent.
  • a range of other solvents can also be used in this step (see, for example, Arbuzov, B.A., Pure Appl . Chem., 1964, 9, 307-335) .
  • the reaction can be carried out at any temperature, and for any time period that achieves the desired results.
  • the reaction is usually carried out at a temperature ranging from -20°C to the boiling temperature of the solvent. It is preferable to limit the access of moisture and oxygen.
  • the reaction time depends upon the structure of the substrate used, the solvent, and the temperature of reaction, and is in general rom 1 to 24 hours .
  • Alkynyl starting materials other that propargyl bromide can be used in this process .
  • Propargyl iodide or propargyl chloride can be substituted for propargyl bromide.
  • 3- butyn-1-bromide will provide a carboranyl- ethylphosphonate, and 4-pentyn-1-bromide will give carboranylpropylphosphonate.
  • appropriately selected homologs of propargyl bromide can be used to prepare any carboranyl (CH 2 ) n P isomer of interest.
  • O,O-dimethylpropargyl- phosphonate is reacted with decaborane in acetonitrile, according to the general reaction scheme described by Heying et al . , Jnor. Chem . 1963, 1089-1092 to provide O,O-dimethyl (o-carboran- 1-yl)methylphosphonate in good yield.
  • the reaction is typically carried out in a Lewis base solvent, e.g., acetonitrile, propionitrile, amine, dialkyl sulfide, cyclic or acyclic ether (tetrahydrofuran, dioxane, diethyl and diisopropyl ether) , or an aromatic solvent as benzene.
  • a Lewis base solvent e.g., acetonitrile, propionitrile, amine, dialkyl sulfide, cyclic or acyclic ether (tetrahydrofuran, dioxane, diethyl and diisopropyl ether)
  • an aromatic solvent as benzene.
  • the reaction can be carried out at any temperature, and for any time period that achieves the desired results.
  • the temperature of reaction generally ranges from room temperature to the boiling temperature of solvent, and the time period of reaction, which depends on the structure of the substrate and reaction conditions, is, in general, from 1 to 24 hours.
  • the target key starting material O-methyl (o- carboran-1-yl)methyl phosphonate is obtained as a triethylamine salt on demethylation of 0,0- dimethyl (o-carboran-l-yl)methylphosphonate using thiophenol and triethylamine in dioxane.
  • a mixture of thiophenol or thiocresol and triethylamine, diisopropylamine or 1, 8-diazabicyclo [5.4] undec-7- ene (DBU base) or other organic base in dioxane or other chemically inert solvent can alternatively be used.
  • 2-mercaptobenzo- thiazole is used in combination with diisopropylamine (see Tetrahedron Lett . , 1988, 29, 5479-5482) .
  • a base should be used that forms a salt of O-methyl (o-carboran-1-yl)methyl phosphonate that is soluble in the organic solvent used. While organic bases are preferred, some inorganic counterions may be used, e.g., cesium (obtained from cesium hydroxide) .
  • the key starting material 0,0-dimethyl (o- carboran-1-yl)methylphosphonate, triethylammonium salt, is reacted with a 5' -(and 2'- or base-, if appropriate) protected nucleoside in the presence of triisopropylbenzenesulfonyl-chloride as the activating agent and 2,4, 6-collidine and 1- methylimidazole.
  • Triisipropylbenzenesulfonyl chloride is an activating agent which activates the borophosphonylating agent.
  • 2, 4, 6-Collidine is a scavenger of the hydrochloric acid generated during reaction.
  • 1-Methylimidazole is a nucleophilic catalyst which additionally activates the borophosphonylating agent.
  • triisipropylbenzenesulfonyl chloride other arylsulfonyl chlorides, or arylsulfonylazolides can be used.
  • 2,4, 6-collidine other organic bases can be used, e.g., di (isopropyl) ethylamine.
  • 1-Methylimidazole can be replaced by other nucleophilic catalysts such as 5-chloro-l-ethyl-2- methylimidazole and 5-nitro-l-methylimidazole .
  • the reaction is typically run in an inert organic solvents, such as a cyclic ether as tetrahydrofuran, a nitrile such as acetonitrile, or a chlorocarbon as dimethylchloride at a temperature ranging from -10°C to boiling temperature of solvent for a time ranging from 5 minutes to 24 hours under anhydrous conditions.
  • an inert organic solvents such as a cyclic ether as tetrahydrofuran, a nitrile such as acetonitrile, or a chlorocarbon as dimethylchloride
  • a 3 ' -O- [O-methyl- (o- carboran-1-yl-methyl)phosphonated] nucleoside is demethylated as described above to provide the triethylamine salt of a 3 ' -O- [ (o-carboran-1-yl- methyl)phosphonated] nucleoside.
  • the above steps can be carried out using a 3'- (and 2'- or base-, if appropriate) protected nucleoside.
  • the reaction conditions should be adjusted.
  • the solubility of a nucleoside bearing a free 5'- hydroxyl group in general is lower than that with a free 3 '-hydroxyl group when the 5'-hydroxyl is protected, and therefore, adjustment of the solvent may be necessary.
  • the triethylamine salt cf the 3' -O- [ (o-carboran- 1-yl-methyl)phosphonated] nucleoside can then be reacted under anhydrous conditions with a 3'-(2'- and base protected, if appropriate) nucleoside to provide a dinucleotide with a 3 ' , 5' -0, 0- [ (o- carboran-1-yl-methyl)phosphonate] linkage.
  • a 5' -ester can be reacted with a 3 '-hydroxyl group of a second nucleoside.
  • Figure 2 is a schematic illustration of a process for the preparation of thymidine- (3 ', 5') - thymidine (o-carboran-1-yl)methylphosphonate using the key starting material O-methyl (o-carboran-1- yl)methyl phosphonate.
  • the process is described in detail in Example 2.
  • Column chromatography was performed on silica gel 60, 230-400 mesh from
  • the crude product was purified by silica gel column chromatography using a stepwise 0-2% gradient of CH 3 OH in CH 2 C1 2 as eluent . Fractions containing 8 were collected, and the organic solvents evaporated to dryness. The residue was dissolved in CH 2 C1 2 and precipitated from hexanes . The precipitate was dried under vacuum yielding 8 as a mixture of two diastereoisomers (0.1 g, 44%) .
  • Oligonucleotides bearing a 3 ' , 5- ' - [0- (carboran-l- yl) alkyl] phosphate, [S- (carboran-l- yl) alkyl] phosphorothioate, or [Se- (carboran-l- yl) alkyl] phosphoroselenoate internucleotide linkage can be conveniently synthesized using a suitable monomer such as 5' -O-monomethoxytritylnucleoside 3 ' - [0- (carboran-l-yl) alkyl] phosphate, [S- (carboran- l-yl) alkyl] phosphorothioate or [Se- (carboran-l- yl) alkyl] phosphoroselenoate as described previously for the oligonucleotides containing 3' ,5'-[(o- carboran-1-yl) alkyl] phosphonate
  • (carboran-l-yl) alkyl refers to (o-carboran-1-yl) (lower alkyl) , and in particular, (o-carboran-1-yl) (lower linear alkyl) .
  • the monomers are prepared by the reaction of a suitably protected nucleoside with a series of new borophosphorylating agents type of O-methyl- [0-
  • the borophosphorylation reaction (synthesis of the specific monomer) proceeds under the conditions described for 5' -O-monomethoxytritylnucleoside 3'- 0-methyl- [0- (o-carboran-1-yl) alkyl] phosphonate however the reaction conditions (activating agent, nucleophilic catalyst, solvent, temperature and reaction time) are adjusted in light of the substrates used.
  • Borophosphorylating agents type of O-methyl- [0- (o-carboran-1-yl) alkyl]phosphate (31) , O-methyl- [S- (o-carboran-1-yl) alkyl]phosphorothioate (36) , and O-methyl- [Se- (o-carboran-1-yl) alkyl] phosphoro ⁇ selenoate (41) are prepared as follows: O -Methyl - [0- (o- carboran- 1 -yl ) alkyl] phospha te
  • a suitable activating agent yielding O,O-dimethyl- (O-alkynyl)phosphate (28) .
  • Another approach to intermediate 28 is based on the reaction of O,O-dimethylchlorophosphate (27) with alcohol (26) in pyridine or other proper solvent.
  • Another approach to (30) is based on reaction of 0,0- dimethylphosphate (25) or O,O-dimethyl ⁇ chlorophosphate (27) with (o-carboran-1-yl) alkylol (32) as described above, (o-Carboran-1-yl) alkylol (32) can be prepared in the reaction of hydroxyl protected alkynol with dodecaborane followed by deprotection of hydroxyl function.
  • Several approaches can be used to prepare the title compound.
  • [ (carboran-1-yl-methyl)phosphonate] linkage after selective deprotection and phosphitylation of its 3' -end with 2-cyanoethyl-N,N-diisopropyl- chlorophosphoramidite, can be used as a building block for the synthesis of longer oligonucleotides bearing one or more alternating (carboran-1- yl)methylphosphonate linkages by automatic synthesis on solid support. See, for example, Applied Biosystems User Bulletin No. 43 1987, Applied Biosystems, Foster City, CA. Oligonucleotides that include one or more 3 ',5'- 0,0- [ (carboran-1-yl-methyl)phosphonate] linkages can also be prepared using solution techniques as known to those skilled in the art .
  • Natural oligonucleotides are synthesized enzymatically on a small scale routinely. Modified oligonucleotides can be also prepared using enzymes (see Lesnikowski, Z. J., Bioorganic Chem, 1993, 21, 127-155) . Enzymatic synthesis of (carboranyl-1- methyl)phosphonate oligonucleotides can be carried out as a modification of the enzymatic synthesis of methylphosphonate oligonucleotide analogues (see Lesnikowski above) .
  • nucleosides with a carboranyl moiety in the base unit have been previously reported. However, it is believed that this is the first disclosure of oligonucleotides that include one or more nucleosides with carboranyl moieties in selected base units. It has been surprisingly discovered that, while useful oligonucleotides can be prepared that contain a carboranyl-containing base in any of the nucleosides, it is preferred that the carboranyl-containing base be located at the 3' or 5' -terminus or in the nucleoside adjacent to the 3' or 5' -terminal nucleoside, or in some combination thereof. Methods for the automated production of oligonucleotides are described above.
  • oligonucleotides with carboranyl-containing base units for a diverse range of applications, all of which are intended to fall within the scope of this invention.
  • Oligonucleotides that contain one or more carboranyl-containing bases can also be prepared using solution techniques as known to those skilled in the art.
  • Example 3 provides a detailed description for the preparation of an oligonucleotide that contains twelve thymine residues, wherein one or more or the thymidine bases contain carboranyl moieties in the 5-position.
  • Example 4 provides detailed physical characterization of the oligonucleotides prepared in Example 3. These examples are merely illustrative, and not intended to limit the scope of the invention.
  • 5' -O-Dimethoxytritylthymidine 3' - (N,N- diisopropyl-2-cyanoethyl)phosphoramidite was obtained from Chem-Impex International (Wood Dale, IL, lot No. 105198) .
  • Thymidine loaded 1 ⁇ M CPG (500 A pore size) columns were purchased from Applied Biosystems (Foster City, CA) .
  • 5- (o- Carboran- 1 -yl ) -2 ' -O- deoxyuridine (CDU) (13, see Figure 4) .
  • oligonucleotides were then cleaved from the support by incubation in concentrated ammonia at room temperature for 1 hour.
  • the deprotected oligonucleotides were purified by HPLC and for selected cases, separated into nido- [nido-1 , 8 -C 2 B ⁇ R (n or 12) ] and closo-forms [ cl oso-1, 2-C 2 B 10 H 12 ] (see Figure 6 and Table 1) .
  • Kane, R.R. Pak, R.H. , Hawthorne, M.F., J. Org. Chem., 1993, 58, 991-992.
  • bufTer B CH 3 CN/H 2 O (50/50) containing 0.05 M TEAA; ' 25 min from 21% B to 23% B; ' 25min from 26% B to 40% B, 5 min to 60% B and 5 min with 60% B; ' 20 min from 30% B to 55% B;
  • Phosphodiesterase I (EC 3 . 1 . 4 . 1) type VIII from Crotalus durissus terrificus venom (lot 119F0730) was purchased from Sigma (St. Louis, MO) .
  • Polyacrylamide was purchased from International Biotechnologies Inc. (New Haven, CT) .
  • Bispolyacrylamide and urea were bought from Fischer Scientific (Fair Lawn, N.J) .
  • Thermal melting curves for the oligomers were determined on a Varian Cary 4 spectrophotometer interfaced to Dell microcomputer.
  • LSIMS mass spectra were recorded on a Finnigan MAT 95 with Cs + gun operating at 13 keV (glycerin matrix) or with VG 70-S spectrometer.
  • CD spectra were recorded on Jasco, J-600 spectropolarimeter interfaced to an IBM computer.
  • Polyacrylamide gel electrophoresis was performed using a BRL apparatus (Gaithersburg, MD) .
  • HPLC analysis of carboranyl-modified dodecathymidylate (19-24) was performed on a Hewlett-Packard 1050 system, with a Whatman Partisphere C 18 5 ⁇ m, 4.7 x 235 mm column. All analyses were performed at room temperature.
  • Polyacrylamide gel electrophoresis PAGE
  • the samples were visualized using standard autoradiography on X-Omat AR film (Eastman Kodak, Rochester, NY) or in the case of unlabeled oligonucleotides by means of UV shadowing.
  • Radiolabeling of dodeca (thymidine phosphates) containing CDU (19-24) were incubated at 37°C in the presence of 0.5 ⁇ L of T4 polynucleotide kinase and 10 ⁇ Ci [ 32 P ⁇ 7] ATP (5000 Ci/mmole) in a 70 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl 2 and 5 mM dithiothreitol . The final volume of reaction mixture was 10 ⁇ L.
  • reaction mixtures were incubated at 92°C for 2 minutes to heat inactivate the enzyme. Thereafter, 10X tracking dye (0.5 % bromophenol blue, 0.5 % xylene cyanol FF, 30% glycerol in water (5 ⁇ l) was added to the reaction mixture and 5 ⁇ l aliquots analyzed by PAGE.
  • 10X tracking dye 0.5 % bromophenol blue, 0.5 % xylene cyanol FF, 30% glycerol in water (5 ⁇ l
  • Tm Mel ting temperature
  • PES (ethane-sulfonic acid) buffer (pH 7.0) containing 100 mM NaCl, and 1 mM EDTA, in amounts to give a 1:1 ratio. Each strand was present at 40 ⁇ M.
  • Molar extinction coefficients e 260 (per base) were calculated as following: 18: 8,150; d(A) 12 :
  • CD spectra were obtained at 10°C in a jacketed cell flushed with nitrogen gas to prevent condensation. Samples were prepared by the addition of concentrated stock solution of oligomers 19-24 to 2.7 mL of 3.75 mM phosphate buffer (pH 7.0) containing 0.5 mM EDTA and 100 mM NaCl . To cuvettes containing the natural or modified d(T) 12 19-24 was added d(A) 12 solution in an appropriate amount to provide a 1:1 ratio. For duplex formation, the samples were heated to 85°C and cooled slowly to room temperature. CD spectra were obtained from 320 to 200 nm for single strands and duplexes.
  • suBsrrruTE SHEET (RULE 26) A difficulty in carboranyl modified oligonucleotide synthesis is the relatively easy transformation of neutral closo-form of boron cage into its nido-counterpart bearing one negative charge under basic conditions, which makes it arduous to synthesize pure closo-compounds in some cases.
  • a colorimetric assay TLC spray for detection of nido- and closo-carboranyl compounds: 30 mg PdCl 2 in 0.5 L of 1% HCl
  • HPLC analysis it was determined that the CDU sample used was contaminated with up to about 5% of nido-compound.
  • nido-isomer may occur during the deblocking of benzoylated hydroxyl functions of CDU under basic conditions.
  • CDU modified oligonucleotides bearing boron clusters in the nido-form are characterized by lower retention time on reverse phase HPLC column (Table 1) . This allows easy separation of the two components.
  • the melting temperature measurements of the separated nido- and closo-CDU oligonucleotides indicated no significant differences in T m for both forms of CDU modification (Table 1) .
  • Circular dichroism (CD) spectra of single stranded (dT) 12 (18) and CDU modified (dT) 12 (19-24) recorded under analogous conditions were almost identical in term of their shapes and molecular ellipticity value (see Figure 8) .
  • the CD spectra of duplexes formed between CDU modified oligothymidylates (20-24) and unmodified (dT) 12 (18) and (dA) 12 showed a reduction in the magnitude of molecular ellipticity at 246 nm which correlated with increased thermal stability of the duplexes ( Figure 9) .
  • Modified oligonucleotides with carboranyl- containing bases may also serve as primers for various polymerases including human immunodeficiency reverse transcriptase.
  • the oligonucleotide 5'-CDUd(T) n (19.1) is characterized by a T m of 28.0°C compared to a T m of 15.2°C for 5'd(T) 6 CDUd(T) 5 (21.1) (Table 1) .
  • Energy minimization results with (dT) 12 (dA) 12 showed that substitution on the 5' end of the (dT) 12 strand gave overall helix energies that were significantly lower than for duplex substituted on the 3' side of (dT) 12 . Substitution on the interior bases of the duplexes caused even larger helix destabilization.
  • T-H The half-life (T-H) of 3'-CDU-, 3 ' ,5' -CDU- and 3 ' CDU 2 -oligonucleotides were determined by kinetic study of their degradation by SVPDE (snake venom phosphodiesterase) . Aliquots of enzymatic reaction (0, 5, 10, 20, 40, 80 and 240 minutes) were analyzed by HPLC. Calculations of half life time were based on the disappearance of 12-mer (or 11- mer in the case of CDU-modified oligos) after standardization with 2' -deoxycytidine used as internal standard. The results are provided in Table 2.
  • the selected compound for in vivo purposes exhibits an aqueous stability under the conditions described below of at least one hour, a serum stability of at least one hour, a cellular uptake of at least 1% of the dose, a cellular washout of at least 1 hour, and a partition coefficient indicative of suitable lipophilicity.
  • Oligonucleotide stabili ty studies .
  • Oligonucleotide stability in human, mouse, and rat serum The stability of the oligomers is determined by incubating the nucleotides at 37°C for the desired period and, after precipitating the protein with cold 60% MeOH/H 2 0 and lyophilizing the supernatant, the sample is resuspended in buffer and an aliquot analyzed by HPLC.
  • a reverse-phase Merck RP-18, 5 mm x 25 cm column is used to determine the stability of the oligonucleotides.
  • the mobile phase is acetonitrile in triethylammonium acetate (or similar system) .
  • An isocratic flow of 1 mL/min is used, and the peaks are monitored using a UV detector set at 260 nm. Cellular uptake studies .
  • glioma cells U251 (2 x 10 6 cells) are suspended in medium containing 10% fetal calf serum and antibiotics, and incubated at 37°C in a 5% C0 2 incubator. The experiment is initiated with addition of 2-10 ⁇ M [ 3 H] -oligomer (specific activity -1,000-2,000 DPM/pmole) , and cells are exposed for 1, 2, 4, 6, 12, and 24 hours to the drug. Medium is removed and the cells are washed 3 times with cold Hank's balanced salt solution.
  • Extraction is performed with addition of 1 mL of 60% cold methanol/water, followed by storage overnight at -70°C. The suspensions are then centrifuged and the supernatants are lyophilized to dryness. The residues are resuspended in 250 ⁇ L of water, and aliquots containing 50-100 ⁇ L are analyzed by HPLC. Quantitation of intracellular oligomer are conducted by HPLC methods developed in our laboratory. A buffer system close to physiological pH is used if needed.
  • the extracts are obtained by centrifugation and removal of the cell pellet.
  • the extracts are lyophili ' zed and stored at -70°C.
  • the material is resuspended in 200 ⁇ L of HPLC buffer and immediately analyzed. Quantitation of intracellular oligomers is conducted as described above .
  • Parti tion coefficient determination Parti tion coefficient determination .
  • oligonucleotide (1 mg/mL) is dissolved in water and then 1 mL octanol is added. The compound is allowed to partition between the two solvents by shaking for 2 hours. The concentration of the drug is determined in different phases by HPLC, as described above .
  • the carboranyl-containing nucleosides and oligonucleotides described herein can be used in boron neutron capture therapy to treat a variety of disorders, and in particular, cancer, for example brain gliomas, melanomas, and breast cancer.
  • BNCT techniques are well known to those of skill in the art, and are described in detail, for example, in Hatanaka et al , Z. Neurol . , vol. 2 * 04, pp. 309-332 (1973) ; Tolpin et al, Oncology, vol. 32, pp. 223- 246 (1975) ; U.S. Patent Nos.
  • a patient in need thereof is treated with an effective amount of one or more to the disclosed compounds and then exposed to neutrons, preferably epithermal neutrons which should not exceed 5 x 10 12 n/cm 2 (total dose) .
  • neutrons preferably epithermal neutrons which should not exceed 5 x 10 12 n/cm 2 (total dose) .
  • a preferred dose of carboranyl-containing nucleoside or oligonucleotide, or combination thereof, for this purpose is 0.01 to 100 mg/kg of body weight in single dose and preferably 0.1 to 20 mg/kg of body weight in a single dose administered intravenously. It may be advantageous to pulse the dosage in order to accumulate the compound in certain cells, such as glioma cells.
  • the compound can be administered at any suitable time, and is typically administered thirty minutes to one hour prior to neutron irradiation.
  • the compounds can be easily evaluated for anti-HIV activity using a variety of methods, for example those disclosed in European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; PCT Publication Nos. WO 92/14729 and WO 92/14743 filed by Emory University, and Schinazi, et al . , Antimicrobial Agents Chemother. , 34, page 1061 (1990) .
  • the ability of the boron cluster nucleosides to inhibit HBV can also be measured by various experimental techniques. A common assay used to evaluate the ability of the disclosed compounds to inhibit the replication of HBV is described in detail in Korba and Gerin, Antiviral Res. 19: 55-70 (1992) . Ability of Boron Cluster Oligonucleotides to effect Site-Directed Mutagenesis
  • SDM Site Directed Mutagenesis
  • a defective nucleic acid or amino acid sequence is produced on transcription or translation of the nucleic acid sequence.
  • the synthetic boron cluster-containing oligonucleotides disclosed herein can be used to effect site directed mutagenesis in vivo or in vitro.
  • a viral, eukaryotic or prokaryotic genome can be mutated by inserting a carboranyl-containing oligonucleotide into a cell wherein the oligonucleotide hybridizes to a nucleic acid sequence in a manner that causes a mutation during transcription or cell division.
  • viral nucleic acids that can be mutated include those in HIV and hepatitis viruses, including HBV.
  • mutagenic oligonucleotides and mutant E. coli strains lacking a DNA repair mechanism can be used to effect mutations of expressed HIV-l reverse transcriptase. Because mutant repair minus DNA polymerases and RT function similarly, it is possible to perform site directed mutagenesis in vivo during HIV-l RNA reverse transcription in order to effect subsequent expression of integrated viral genome. Expression of the mutated RT gene at the further stages of virus replication cycle results in production of unfunctional enzyme which in turn can inhibit viral replication. Mutations in other target areas of HIV can also be generated as desired using the appropriate sequence .
  • Example 6 describes the in vivo mutation of the RT gene within the YMDD motif, incorporated into a proper vector in E.
  • the mutagenic oligonucleotide DNA target region is the conserved YMDD motif (nucleotides 5' 2678 to 3' 2689) of HIV- 1 RT.
  • the mutagenic oligonucleotides used were 5' -AATACATGGA(CDU)GATTTGTAT-3' and 5' -AATACATGG (CDU) (CDU) GATTTGTAT-3 ' .
  • the oligonucleotides were synthesized using ⁇ - cyanoethyl phosphoramidite chemistry and an automated DNA synthesizer. Each contains one or two modified nucleosides [ (5- (o-carboran-1-yl) -2' - deoxyuridine (CDU)] .
  • the restriction enzymes used for the cloning of the reverse transcriptase (RT) gene were selected to contain the active site of RT and to ensure unique restriction sites for the cloning of mutants back into pBRU.
  • the restriction enzymes Sac 1 (base 228) and Sal 1 (base 5367) were used to place the desired fragment into the phagemid pALTER-1. This plasmid was supplied with the Altered Sites in vi tro Mutagenesis System ( Promega Corp., Madison, Wl. 53711-5399) .
  • the construct was transformed into JM109 (endAl, recAl, gyrA96, thi, hsdR17 (rk “ , m k + ) , relAl, supE44, lambda " , ⁇ (lac-proAB) , [F' , traD36, proA + B + , lacI q Z ⁇ M15] using the method of CaCl 2 transformation as described (Sambrook, J. , Fritsch, E.F., Maniatis, T., 1989, Molecular Cloning. A Laboratory Manual, (Nolan, C, Ed.) Cold Spring Harbor Laboratory Press) .
  • the culture was then infected with R408 and M13K07, helper phages, at a multiplicity of infection (m.o.i.) of 10.
  • the cultures were then shaken at 200 rpm overnight at 37°C.
  • the next morning the culture was harvested by spinning at 12,000 rpm for 15 minutes, and the supernatant was removed to a new tube.
  • the supernatant was then spun again at 12,000 rpm for 15 min to remove any remaining cells or debri.
  • the phage was then precipitated by adding 0.25 volumes of a polyethylene glycol (PEG) solution (3.75 M ammonium acetate- 20% polyethylene glycol; MW 8,000) .
  • PEG polyethylene glycol
  • the sample was then placed on ice for 30 min, followed by centrifugation at 12,000 x g for 15 min.
  • the pellet was thoroughly drained by inverting the tube on a paper towel for 2-3 min.
  • the pellet containing phage was then resuspended using 400 ⁇ L of TE buffer [10 mM Tris-HCl (pH 8.0, 1 mM EDTA)] .
  • An equal volume of chloroform: isoamyl alcohol (24:1) was added directly to the resuspended phage in order to lyse the particle and to remove any excess PEG solution.
  • the mixture was inverted several times and spun at 12,000 x g for 5 min.
  • the upper aqueous phase was removed to a new tube and an equal volume of TE saturated phenol :chloroform:isoamyl alcohol (25:24:1) was added. The solution was then inverted several times and spun at 12,000 x g for 5 min. This step was repeated until no visible interface could be detected. From the final phenol :chloroform:isoamyl alcohol extraction, the aqueous phase was removed to a new clean eppendorf tube. Finally, 0.5 volumes of 7.5 M ammonium acetate and 2 volumes of ice cold 95 % EtOH was added to the solution. The solution was placed at -70°C undisturbed.
  • the sample was spun at 14,000 x g for 30 minutes followed by two washes with 70 % EtOH at 14,000 x g for 15 minutes each. The sample was then dried in a speed vac at room temperature for 10 min. Samples were then resuspended in 20 ⁇ L of dH 2 0 and spot checked on ethidium stained 0.8 % agarose gel run at 45 volts for approximately 1.5 hour.
  • AATACATGG (CDU) (CDU) GATTTGTAT -3 ' The modified oligonucleotides were synthesized using an Applied Biosystems 391 DNA synthesizer. Columns loaded with controlled pore glass functionalized with 5'-0- dimethoxytrityl thymidine (1 mM) were utilized as solid support. All 5' -dimethoxytrityl 3'- phosphoramidite derivatives were prepared as 0.09 M solution in anhydrous CH 3 CN. Elongation of oligonucleotides was performed using standard ⁇ - cyanoethyl 1 mM DNA synthesis cycle ⁇ Applied Biosystems USER Bulletin No.
  • Oligonucleotides were then deprotected and cleaved from the support by overnight incubation in concentrated NH 4 OH at room temperature. The oligonucleotides were then purified using OPCTMcartridge (Applied Biosystems, Foster City, CA) and their purity was checked by HPLC as described below.
  • AATACATGG(CDU) (CDU) GATTTGTAT-3' and a control, located on the putative catalytic active site for HIV-l RT (5' -2676 to 3'- 2696) were 5' phosphorylated using T4 polynucleotide kinase.
  • the reaction was terminated by heating to 70°C for 10 min.
  • the mutagenesis annealing reaction was performed by adding 0.05 pmol of pHIVALT-1 ssDNA, 0.25 pmol ampicillin repair oligonucleotide (supplied in the kit) , 1.25 pmol of the 5' phosphorylated mutagenic oligo, and an annealing buffer supplied in the kit in a final volume of 20 ⁇ L.
  • control oligonucleotide reactions for this experiment were done separately along with an added reaction that contained ampicillin repair oligo but no mutagenic oligo 5' -AATACATGGA(CDU) GATTTGTAT-3 ' and 5 ' -AATACATGG (CDU) (CDU) GATTTGTAT-3' and control unmodified.
  • the reactions were then heated to 70°C for 5 min and allowed to cool to room temperature over a 15-20 min period.
  • the annealing reactions were placed on ice and the following reagents were added: 3 ⁇ L 10X synthesis buffer (supplied in kit) , 1 ⁇ L T4 DNA polymerase (10 U/ ⁇ L ) , 1 ⁇ L T4 DNA ligase ( 2 U/ ⁇ L ) , and 5 ⁇ L sterile dH 2 0. The 30 ⁇ L reaction was incubated at 37°C for 90 min to perform second strand synthesis and ligation.
  • the purified plasmid DNA was resuspended in 20 ⁇ L of sterile dH ; 0 and transformed into competent JM109 as described above and plated on LB agar plates supplemented with 100 ⁇ g/mL ampicillin.
  • RT-MT4 (5'- CAATGAGACACCAGGG-3' ) located 5'- 2539 to 3'- 2554 of HIVBRU
  • RT-MT7RC 5' -GTCATTGACAGTCCAGCTGTC- 3'
  • the primers are located on opposite sides of the active site of RT and would give sequence verification on both strands of the plasmid.
  • the oligonucleotides of the present invention can be used as probes in a variety of diagnostic techniques, including Magnetic Resonance Imaging (MRI) .
  • MRI Magnetic Resonance Imaging
  • MRI is a noninvasive technique used to detect the presence and location of tumors in a patient.
  • cancer cell specific boronated compounds are administered to a patient which concentrate in the cancerous tissue.
  • the MRI instrument is capable of detecting and locating regions of abnormal concentrations of boron. By indicating the regions having high relative concentrations of boron, MRI establishes the presence and location of the tumors.
  • Another diagnostic application of the oligonucleotides of the present invention is their use as molecular probes.
  • the oligonucleotide is used to detect the presence of complementary sequences of DNA or RNA in a sample by hybridization according to standard techniques.
  • the probes can be used in a Southern blotting and Northern blotting assay, the details of which are known. See, e.g., R. Old and S. Primrose, Principles of Gene Manipulation, 8-10 (3d Ed. 1985) .
  • the boron atom serves as a radiolabel, though it is not itself radioactive until exposed to neutron radiation.
  • 10 B absorbs a neutron and forms unstable n B, which rapidly decays and releases an alpha particle and gamma radiation, thus providing a detectable signal .
  • the techniques involved in the generation of the alpha particle are known. See, e.g., A. Soloway, Borax Rev. 3, 7- 9 (1988) .
  • Reaction conditions for hybridization of an oligonucleotide probe or primer to a nucleic acid sequence vary from oligonucleotide to oligonucleotide, depending on factors such as oligonucleotide length, the number of G and C nucleotides, and the composition of the buffer utilized in the hybridization reaction.
  • Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25°C below the melting temperature of a perfectly base-paired double- stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions. Chapter 11 of the well-known laboratory manual of Sambrook et al .
  • the boron content in 10 6 cells can be obtained by lysing the cells to be analyzed, and then proceeding as described above. This procedure can be easily adapted by one of skill in the art for diagnosis using the boron containing probes .
  • Oligonucleotides of the present invention which are capable of binding to polyribonucleic acid or polydeoxyribonucleic acid are useful as antisense agents in the same manner as conventional antisense agents. See generally Antisense Molecular Biology and S-oligos, Synthesis 1 (Oct. 1988) (published by Synthecell Corp., Rockville, Md.) ; 2 Discoveries in Antisense Nucleic Acids (C. Brakel and R. Fraley eds . 1989) ; Uhlmann, et . al . , "Antisense
  • Antisense agents of the present invention may be used by constructing an antisense agent which is capable of selectively binding to a predetermined polydeoxyribonucleic acid sequence or polyribonucleic acid sequence to a " cell containing such sequence (e.g., by adding the antisense agent to a culture medium containing the cell) so that the antisense agent is taken into the cell, binds to the predetermined sequence, and blocks transcription, translation, or replication thereof.
  • the requirements for selective binding of the antisense agent are known (e.g., a length of 17 bases for selective binding within the human genome) .
  • the carboranyl-modified nucleosides and oligonucleotides and any combinmation thereof can be administered to humans in an effective amount for any of the purposes described herein.
  • the active material can be optionally be administered as a pharmaceutically acceptable derivative or salt, or optionally, in combination with a pharmaceutically acceptable carrier or diluent.
  • the active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
  • the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to achieve the desired therapeutic result without causing serious toxic effects in the patient treated.
  • compounds with a therapeutic index of at least 2, and preferably at least 5 or 10 are acceptable.
  • the therapeutic index is defined as the IC 50 /EC 50 , wherein EC 50 is the concentration of compound that inhibits the growth * by 50% of the diseased cells and IC 50 is the concentration of compound that is toxic to 50% of the otherwise healthy target cells.
  • Cellular toxicity can be measured by direct cell counts, trypan blue exclusion, or various metabolic activity studies such as 3 H-thymidine incorporation, as known to those skilled in the art.
  • a preferred dose of the active compound for all of the above-mentioned conditions will be in the range of 0.01 to 1000 mg/kg of body weight and preferably 0.1 to 20 mg/kg of body weight in a single dose per day.
  • the effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.
  • the compound is conveniently administered in unit any suitable dosage form, including but not limited to one containing 5 to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit dosage form.
  • a oral dosage of 50-1000 mg is usually convenient .
  • the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.01 to 100 ⁇ M, preferably about 0.1 to 40 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.1 to 2% solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient .
  • the active compound is administered in an intravenous solution with a dose ranging from 1 mg/kg to 20 mg/kg.
  • the active compound is administered in a pharmaceutical composition for oral delivery that protects the compound from the acid environment of the stomach, for example, an enteric coating.
  • concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that, when using the compound to treat a disease, dosage values will vary depending on the severity of the condition to be alleviated.
  • compositions should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compound or a pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active compound or pharmaceutically acceptable derivative or salt thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or other antivirals, including other nucleoside anti-HIV compounds .
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS) .
  • PBS physiological saline or phosphate buffered saline
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Scios-Nova Pharmaceuticals, Inc.
  • Liposomal suspensions are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (which is incorporated herein by reference in its entirety) .
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or triphosphate derivatives are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL YES
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL YES
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention se rapporte à des nucléosides et à des oligonucléosides contenant du carboranyle, destinés à être utilisés en boroneutrothérapie (BNCT) ainsi qu'à d'autres fins thérapeutiques et diagnostiques.
EP95904208A 1993-12-02 1994-12-02 Nucleosides et oligonucleosides contenant des amas de bore Expired - Lifetime EP0731808B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00203565A EP1113020B1 (fr) 1993-12-02 1994-12-02 Nucléosides et oligonucléosides contenant des amas de bore

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Publication number Priority date Publication date Assignee Title
US6583122B2 (en) 1993-12-02 2003-06-24 Emory University Nucleosides and oligonucleotides containing boron clusters

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JP2009067777A (ja) 2009-04-02
DE69435247D1 (de) 2009-12-10
AU1299195A (en) 1995-06-19
EP1113020B1 (fr) 2009-10-28
WO1995015333A1 (fr) 1995-06-08
EP1113020A2 (fr) 2001-07-04
CA2177952A1 (fr) 1995-06-08
DE69427705D1 (de) 2001-08-16
US20020160969A1 (en) 2002-10-31
AU685892B2 (en) 1998-01-29
JPH10505318A (ja) 1998-05-26
JP4344959B2 (ja) 2009-10-14
EP0731808A4 (fr) 1997-04-02
US6180766B1 (en) 2001-01-30
ES2161279T3 (es) 2001-12-01
CA2177952C (fr) 2008-04-29
DE69427705T2 (de) 2002-07-04
EP0731808B1 (fr) 2001-07-11
CA2617836A1 (fr) 1995-06-08
EP1113020A3 (fr) 2003-04-16

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